Therapeutic Mechanism And Clinical Application Of Chinese Herbal Medicine Against Diabetic Kidney DiseaseⅡ

The inhibition of oxidative stress and inflammation 

Oxidative stress and inflammation drive the development of DKD, and several novel mediators are involved in such process, including tonicity-responsive enhancer-binding protein, apoptosis signal-regulating kinase 1, serine/threonine protein kinase 25, and receptor activator of NF-κB (Chen et al., 2017a; Chen et al., 2017b; Liles et al., 2018; Chen et al., 2019b; Cansby et al., 2020; Choi et al., 2020; Ke et al., 2021; Liu et al., 2022b). Additionally, superoxide dismutase (SOD), glutathione peroxidase, and malondialdehyde are considered biomarkers of oxidative stress to evaluate the effects of Chinese herbal medicine on DKD treatment (Zhou et al., 2022). Even mechanistic studies concerning DKD progress develop a lot, mechanistic studies concerning Chinese herbal medicine against DKD are relatively hysteretic, in that most the Chinese herbal medicine alleviates oxidative stress and inflammation in DKD by modulating NF-κB and Nrf2 pathways (Figure 3; Table 2). Curcumin significantly ameliorates albumin/protein urea and increased creatinine clearance in STZ-induced DKD rats, which involves the activation of Nrf2 and the inhibition of NF-κB, NADPH oxidase and significant up-regulation of the protein kinase Cβ II (PKCβII)/p66Shc axis (AlTamimi et al., 2021). The inhibition of NLRP3 inflammasome activity and the downregulation of kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin are also therapeutic targets of curcumin to suppress oxidative stress against DKD progression (Lu et al., 2017; Ghasemi et al., 2019). The combination of curcumin with antihyperglycemic agents exerts stronger effects against diabetic complications by maintaining Nrf2 pathway homeostasis (Xie et al., 2021). Fructus Arctii attenuates proteinuria in patients with diabetes, and apigenin, a lignan extracted from F. Arctii, reduces proteinuria and podocyte injury in diabetes mouse models. Arctigenin enhances protein phosphatase 2 A (PP2A) activity to alleviate p65-mediated inflammation in vivo and blocks the endoplasmic reticulum (ER) stress signal transduction pathway to suppress apoptosis in high glucose-induced HK2 cells (Zhang et al., 2019a; Zhong et al., 2019). Specific deletion of PP2A in podocytes weakens the efficiency of arctigenin, indicating PP2A as the therapeutic target of arctigenin (Zhong et al., 2019).

Beyond NF-κB and Nrf2 pathways, the suppression of mitogen-activated protein kinase (MAPK)-mediated inflammatory signaling pathway is the therapeutic target of baicalin. Baicalin attenuates diabetic conditions, proteinuria, and renal histopathological changes, and alleviates oxidative stress and inflammation in DKD animal models via Nrf2 and MAPK pathways (Ma et al., 2021). Ellagic acid alleviates high glucose-induced mesangial cell injury and inflammation in a concentration-dependent manner, and the underlying mechanisms involve the activation of PI3K/Akt signaling pathway and the suppression of forkhead box transcription factor 3a (FoxO) 3a during DKD (Lin et al., 2021). Astragaloside IV alleviates podocyte injury and delays DKD progression in db/db mice by suppressing NLRP3 inflammasome-mediated inflammation (Feng et al., 2021). Yi Shen Pai Du Formula inhibits oxidative stress, inflammation, and EMT to delay DKD progression in db/db via activating the Nrf2 pathway (Zhang et al., 2021a). Tangshan Formula, a Chinese formulation, exerts beneficial effects against DKD by modulating thioredoxin-interacting protein (TXNIP)- NLRP3-gasdermin D (GSDMD) axis-mediated pyroptosis in STZ-induced rat model and AGE-induced HK-2 cells (Li et al., 2020).

The modulation of mitochondrial dysfunction 

PGC-1α is a prominent modulator of mitochondrial biogenesis and an attractive therapeutic target in DKD treatment (Li and Susztak, 2016; Long et al., 2016; Trembinski et al., 2020), and several Chinese herbal medicines and their active compounds exert renoprotection against DKD by modulating PGC-1α (Figure 4; Table 2). Metabolomic research proves that the abnormality in mitochondrial fuel usage and mitochondrial dysfunction occurs in patients with DKD, and berberine, the main active compounds of Rhizoma copies and Cortex phelloderm, modulates PGC-1α to alleviate mitochondrial injury in direct and indirect pathways. Berberine facilitates mitochondrial energy homeostasis and fatty acid oxidation by directly activating the PGC-1α signaling pathway and protects glomerular podocytes via inhibiting dynamin-related protein 1-mediated mitochondrial fission and dysfunction in db/db mice model and cultured podocytes (Qin et al., 2019; Qin et al., 2020). Berberine activates the AMPK pathway to upregulate PGC-1α which reduces fatty acid oxidation, and lipid deposition and protects mitochondria to mitigate diabetic renal tubulointerstitial injury (Rong et al., 2021). Additionally, berberine modulates mitochondrial reactive oxygen species (ROS) generation by stimulating the positive feedback loop of CCAAT enhancer binding protein beta (C/EBPβ)/growth arrest-specific 5 (Gas5)/miR-18a-5p (Xu et al., 2021a). The ethanol extract of Rhodiola rosea exerts beneficial protection in the STZinduced model in the early nephropathy in type 2 diabetic rats (Wang et al., 2013). 

Click to cistanche beneficial for kidney disease

Further study identifies salidroside as a major active compound of R. rosea to treat DKD. Salidroside markedly improves renal structures and reverses the downregulation of nephrin and podocin in patients with DKD. Mechanistically, the salidroside treatment promotes mitochondrial DNA copy and electron transport chain proteins by enhancing Sirt1 and PGC-1α expression in STZ-induced mice (Xue et al., 2019). Salidroside also suppresses oxidative stress and ECM accumulation by modulating the TXNIP-NLRP3 inflammasome pathway (Wang et al., 2017), and reduces proteinuria by attenuating caveolin-1 phosphorylation and albumin transcytosis across glomerular endothelial cells (Wu et al., 2016). Resveratrol, a potent Sirt1 agonist, attenuates podocyte damage in diabetic mice by suppressing mitochondrial oxidative stress (Zhang et al., 2019c), while 4-O-methyl honokiol, isolated from Magnolia stem bark, protects against STZ-induced DKD by activating AMPK/PGC1α/carnitine palmitoyltransferase 1B (CPT1B)-mediated fatty acid oxidation and Nrf2/SOD2-mediated anti-oxidative stress (Ma et al., 2019). Notably, Tangshan Formula and its active compounds Morningside enhance renal cholesterol efflux to ameliorate tubular epithelial injury in db/db mice by activating PGC-1α-liver X receptor (LXR)-ATP-binding cassette transporter A1 (ABCA1) pathway (Liu et al., 2018; Gao et al., 2021a), indicating a promising candidate for DKD treatment.

Other potential therapeutic targets are also proved, including PTEN-induced putative kinase 1 (PINK1) and peroxisome proliferator-activated receptor (PPAR) (Figure 4; Table 2). Several studies show the renoprotective effects of astragaloside II and astragaloside IV, active compounds of Astragalus membranes, referring to the improvement of mitochondrial dysfunction. Astragaloside II and astragaloside IV ameliorate albuminuria in DKD animal models and prevent podocyte injury from high glucose (Su et al., 2021a; Xing et al., 2021; Zang et al., 2021). Astragaloside II suppresses mitochondrial dysfunction of podocyte injury via upregulating PINK1 and Parkin, and astragaloside IV inhibits cytochrome c release and mitochondrial membrane potential to protect podocytes against DKD (Su et al., 2021a; Zang et al., 2021). 

Astragaloside II also mitigates podocyte apoptosis against DKD via suppressing transient receptor potential channel 6- mediated Ca2+ influx, and astragaloside IV activates the PPARγKlotho-FoxO1 pathway to alleviate podocyte apoptosis (Xing et al., 2021; Zang et al., 2021). Ginsenoside Rb1 combines with aldose reductase to alleviate mitochondrial damage and podocyte apoptosis thereby delaying the progression of DKD (He et al., 2022). Quercetin, an active compound from Panax notoginseng, antagonizes glucose fluctuation-caused kidney injury by inhibiting aerobic glycolysis via hypoxia-inducible factor-1 alpha (HIF-1α)/miR-210/ISCU/FeS pathway in glomerular mesangial cells (Xu et al., 2021b). Andrographolide isolated from Andrographis paniculate suppresses mitochondrial ROS-mediated NLRP3 inflammasome activation to ameliorate mitochondrial dysfunction during DKD (Liu et al., 2021b). Chinese herbal formula Huangqi-Danshen decoction delays DKD progress by suppressing PINK1/Parkin-mediated mitophagy (Liu et al., 2020b). Additionally, the signal transducer and activator of transcription 3 is also a potential therapeutic target to modulate mitochondrial homeostasis through SDF1α/CXCR4 pathway to ameliorate renal tubular injury in DKD (Zhang et al., 2020b). 

The regulation of gut microbiota and related metabolism 

Emerging evidence have confirmed the relationship between the dysfunction of gut microbiota and related metabolism and the progression of DKD indicating the importance of the kidney-gut axis (Winther et al., 2020; Yang et al., 2021). According to 16S rRNA sequencing and metabolomic results, the gut microbiota structure, phenylalanine, and tryptophan metabolic pathways are significantly altered in patients with DKD (Zhang et al., 2021b). Phenyl sulfate is a gut microbiota-derived metabolite, and its level increases with the progression of diabetes in a rat models. Phenyl sulfate contributes to albuminuria and could be used as a biomarker for DKD (Kikuchi et al., 2019). Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite, and serum TMAO closely relates to and mediates impaired renal function (Winther et al., 2019). As an important component of innate immunity, mitochondrial antiviral signaling protein (MAVS) maintains intestinal integrity. DKD contributes to the impairment of MAVS signaling in the kidney and intestine thus leading to disrupted homeostasis, indicating that maintaining intestinal homeostasis may function as a novel therapeutic approach for DKD treatment (Linh et al., 2022). 

Notably, gut microbiota and related metabolism are the potential therapeutic target of Chinese herbal medicine against DKD (Table 2). Punicalagin isolated from Punica granatum reshapes gut microbial ecology, reverses gut barrier dysfunction, and reduces serum lipopolysaccharide and diamine oxidase levels to delay DKD progression (Hua et al., 2022). Functioning as a prebiotic, the polysaccharide extracted from M. Cortex reconstructs gut microbiota, improves intestinal barrier function, ameliorates serum proinflammatory mediators, and upregulates short-chain fatty acid levels by controlling Lactobacillus and Muribaculaceae_unclassified abundance in the gut of DKD rat model (Zhang et al., 2022). Qing-Re-XiaoZheng formula reverses gut dysbiosis and inhibits the generation of gut-derived LPS, and suppresses DKD-related inflammation by reducing TLR4 and NF-κB expression in DKD mouse model (Gao et al., 2021b). 

QiDiTangShen granules exert good efficacy in alleviating proteinuria in the DKD mice model, and the underlying mechanisms involve the modulation of the gut microbiota-bile acid axis via the farnesoid X receptor (Wei et al., 2021). Shenyan Kangfu tablet, a prescription of traditional Chinese medicine, attenuates stimulated blood glucose and glycosylated hemoglobin (HbA1c) levels and alleviates renal dysfunction and inflammation in db/db mice. The underlying mechanisms refer to suppressed renal inflammatory signaling cascades and improved intestinal microbiota via elevated Firmicutes and reduced Bacteroidetes abundance (Chen et al., 2021). San-Huang-Yi-Shen capsule exhibits beneficial effects against DKD in clinics. The mechanism involves the improvement of gut microbiota by modulating arginine biosynthesis, tricarboxylic acid (TCA) cycle, tyrosine metabolism, and arginine and proline metabolism (Su et al., 2021b). In addition to the above-mentioned mechanisms, Tangshan Formula attenuates diabetic renal injury and inflammation by regulating gut microbiota to reduce lipopolysaccharide and indoxyl sulfate levels (Zhao et al., 2020).

Clinical application of Chinese herbal medicine in diabetic kidney disease treatment Even though a lot of work engages to elucidate the underlying mechanisms of Chinese herbal medicine, the lack of high quality evidence from clinical trials significantly hinders the application of Chinese herbal medicine worldwide. Here, we summarize randomized clinical trials (RCTs) of Chinese herbal medicine against DKD to highlight its beneficial efficacy and found that Chinese herbal medicine can be used as the primary and additional treatment regimen for DKD in clinics. Single-use of Chinese herbal medicine shows beneficial efficacy on DKD treatment. A retrospective study reports the beneficial efficacy of traditional Chinese medicine, Shenzhuo formula, on patients with DKD. The changes in estimated GFR (eGFR), creatinine clearance, serum creatinine, blood urea nitrogen, albuminuria, HbA1c, blood pressure, and lipid profile are observed. 

Compared with the baseline, serum creatinine significantly decreases, and the estimated glomerular filtration rate (eGFR) and creatinine clearance increase after intervention at 1, 3, 6, 9, 12, and 18 months. Shenzhuo formula also reduces HbA1c, lipid levels, and blood pressure (Tian et al., 2015). A multicenter, parallel-control, open-label, RCT investigates the effect of Zicuiyin decoction on DKD treatment, and the primary outcome is the change of eGFR. Zicuiyin decoction significantly increases eGFR and decreases serum creatinine to alleviate DKD via correcting gut microbiota dysbiosis (Liu et al., 2022a). Additionally, a single-blind, randomized, controlled preliminary study explores the efficacy of acupressure at Sanyinjiao for DKD treatment, and the primary outcome measure is the UACR or logarithmic transformed UACR (log-UACR) changes. The difference in UACR and log-UACR before and after the study was higher in the Sanyinjiao group than in the sham groups, and the acupressure at Sanyinjiao for 8 weeks helps to decrease albuminuria in patients with early DKD indicated by eGFR and HbA1c (Chuang et al., 2020). The combined therapy of Chinese herbal medicine and ARB/ ACEI also exhibit favorable efficacy in DKD treatment. Huangkui capsule from traditional Chinese medicine is made from the ethanol extract of flowers in Abelmoschus manihot. A multicenter randomized double-blind parallel controlled clinical trial is designed to evaluate the effect of combined Huangkui capsule and irbesartan treatment on DKD, and the primary outcomes are changed values of albumin-to-creatinine ratio from baseline after treatment. 

Combined Huangkui capsule and irbesartan treatment 

exhibits beneficial effects on alleviating albuminuria in patients with type 2 diabetes and DKD (Zhao et al., 2022). Additionally, single Huangkui capsule therapy and combined Huangkui capsule and losartan therapy exert favorable therapeutic effects against primary glomerular disease in a prospective, multicenter randomized controlled clinical trial (Zhang et al., 2014). A multicenter double-blinded randomized placebo-controlled trial shows that accompanied by conventional ARB or ACEI treatment, Tangshan Formula treatment for continuous 24 weeks exhibits beneficial efficacy compared with placebo on decreasing proteinuria and improving eGFR in DKD patients with macroalbuminuria (Li et al., 2015). In this study, primary outcomes are urinary protein level, measured by urinary albumin excretion rate (UAER) for participants with microalbuminuria, and 24-h urinary protein for participants with macroalbuminuria. Except for UAER, Tangshan Formula treatment exhibits favorable effects on other primary outcomes. 

Further investigation indicates that urinary liver-type fatty acid binding protein is identified as the biomarker for the severity of DKD and the effects of Tangshen Formula against DKD (Yang et al., 2016a). The ongoing RCT of Tangshen Formula aims to investigate its effectiveness and safety in treating type 2 DKD patients with macroalbuminuria (Yan et al., 2016). The meta-analysis of RCTs shows the satisfied efficacy of combined Tripterygium wilfordii Hook, tripterygium glycosides, Ophiocordyceps sinensis, or Jinshuibao with conventional ACEI or ARB treatment against DKD (Luo et al., 2015; Lu et al., 2018; Zhang et al., 2019b; Ren et al., 2019; Wu et al., 2020; Yang et al., 2020). Notably, several RCT protocols have been designed to provide solid evidence for combined Chinese herbal medicine and conventional ARB or ACEI therapy against DKD. 

An assessor-blind, parallel, pragmatic randomized controlled clinical trial registered in Hong Kong has been engaged to evaluate the effectiveness of add-on astragalus in clinics. This trial plans to enroll 181 patients with type 2 diabetes, stage 2-3 CKD, and macroalbuminuria who receive 48 weeks of add-on astragalus or standard medical care (Chan et al., 2021). A double-blind, placebo-controlled, randomized trial is designed to explore the efficacy and safety of combined Liuwei Dihuang pills with conventional metformin and ARB therapy against DKD for 4 weeks of treatment and 12 weeks follow-up, and 24 h urinary protein levels from the baseline to the end of the treatment phase is the primary outcome (Liao et al., 2020). A prospective, single-center RCT aims to investigate the efficacy and safety of combined Tripterygium glycosides and ARB therapy for DKD treatment. The primary endpoint is 24 h proteinuria decreased level after treatment for 48 weeks (Lengnan et al., 2020).

Conclusion and perspectives 

DKD is the leading cause of CKD and ESRD worldwide. Although the drug development of RAAS and SGLT2 inhibitors has evolved, a large proportion of DKD patients still need dialysis and renal transplantation. The beneficial efficacy of Chinese herbal medicine in clinical application attracts a lot of attention as an alternative therapy. DKD progression is normally considered irreversible, while Chinese herbal medicine gives us hope. Reliable evidence from RCTs shows that Tangshen Formula and T. wilfordii Hook extract significantly reduce proteinuria and elevate eGFR compared with ARB or ACEI (Ge et al., 2013; Li et al., 2015; Liu et al., 2021a). Chinese herbal medicine normally targets multiple and synergetic targets to alleviate DKD due to multiple active compounds, including the improvement of metabolism regulation, the inhibition of oxidative stress and inflammation, the modulation of mitochondrial dysfunction, and the regulation of gut microbiota and related metabolism. Notably, we notice that many Chinese herbal medicines synergistically target multiple key factors and pathways to ameliorate DKD, including icariin, catalpol, C. tinctoria, salidroside, and 4-O-methyl honokiol (Table 2). 

These promising candidates highlight the advantage that synergistically targeting multiple key factors and pathways is an important strategy to facilitate drug development for DKD treatment. These also highlight the importance and urgency to discover and identify the novel therapeutic target. Another advantage of Chinese herbal medicine is its clinical experience for thousands of years in east Asia. Chinese herbal medicine has been still used to prevent and treat DKD today, and many high-quality clinical pieces of evidence confirm the efficacy of combined Chinese herbal medicine and conventional western medicine, such as Tangshen Formula, Xiaokeyinshui formula, and T. wilfordii, which provide an alternative strategy for DKD treatment. However, some limitations hinder the recognition and extensive use of Chinese herbal medicine beyond east Asia. One is the lack of high-quality evidence to identify the therapeutic mechanisms of Chinese herbal medicine. Most research reported the modulation of Chinese herbal medicine on common mechanisms rather than specific and targeted mechanisms via high throughput analysis. Considerable work needs to be done to identify the targeted mechanism of Chinese herbal medicine on DKD treatment. 

The multiple active compounds of Chinese herbal medicine contribute to the beneficial efficacy to alleviate DKD but also result in difficulty to control the quality of Chinese herbal medicine. The identification of active compounds and the establishment of a corresponding quality control system is necessary to ensure the efficacy and safety of Chinese herbal medicine. Additionally, strict and standardized toxicological research is essential to ensure acceptable side effects. The proper dosage and duration of Chinese herbal medicine should be investigated in preclinical and clinical trials to ensure favorable efficacy. The lack of high-quality evidence from clinical medicine significantly hinders the extensive use of Chinese herbal medicine, and more efforts are needed to solve this problem. Fortunately, RCTs and mechanism studies of some Chinese herbal medicines have been completed and some are ongoing. For example, the clinical efficacy of Tangshen Formula has been investigated by RCTs, and Tangshen Formula exerts better efficacy in reducing albuminuria and elevating eGFR (Li et al., 2015). 

Meanwhile, mechanism studies show that Tangshen Formula synergistically targets multiple key factors or pathways to ameliorate DKD via modulating pyroptosis, enhancing renal cholesterol efflux, reshaping gut microbiota, and suppressing inflammation (Liu et al., 2018; Li et al., 2020; Zhao et al., 2020). Tangshan Formula sets up a good example that provides reliable evidence for clinical trials and therapeutic mechanisms. Given that many Chinese herbal medicines have yet to be investigated using a modern pharmacological approach, we anticipate many of them could be completed in the future. Chinese herbal medicine has its advantages in treating DKD, including multiple therapeutic targets and rich clinical experience. New guidelines concerning Chinese herbal medicine against DKD are needed to assure safety and efficacy to amplify its application in treating DKD worldwide.

for more information: Ali.ma@wecistanche.com